Development of Broad-range and Specific 16S rRNA PCR for Use in Routine Diagnostic Clinical Microbiology

Hyun-Chul Kim(김현철),Yun-Tae Kim(김윤태),Hyogyeong Kim(김효경),Sanghoo Lee(이상후),Kyoung-Ryul Lee(이경률),Young-Jin Kim(김영진) 한국생명과학회 2014 생명과학회지 Vol.24 No.4

16S rRNA gene PCR법은 환자 검체로부터 병원성 미생물을 검출 및 동정에 사용되어진다. 본 연구는 대량의 임상미생물 진단을 위해 bacterial 16S rRNA 부위 유전자 서열을 이용하여 광대한 범위와 높은 특이도를 가지는 primer을 포함한 PCR법을 개발하였다. 10개 표준 균주 16S rRNA 보존 부위의 유전자 서열을 기반으로 primer set를 구축하였다. 98명 환자 검체에서 임상 미생물을 분리하였다. 98개 균주는 phenotypic 방법을 이용하여 확인하고, 개발된 primer set와 universal primer set를 이용한 PCR법으로 확인하였다. 획득한 PCR 산물은 forward primer, reverse primer, 그리고 자동화 DNA 분석기를 이용하여 각 균주의 16S rRNA 유전자 서열을 분석 및 확인하였다. 본 연구에서 개발된 primer set와 universal primer set의 임상미생물 검출에 대한 효율성을 평가하였고, 또한 phenotypic 방법과 분자생물학적 방법을 비교했다. 분리된 98개 균주를 대상으로 개발된 primer set로 16S rRNA PCR을 진행하여 778 bp 크기의 단일밴드로 증폭 되었음을 확인했다. 총 98개중 94개 균주(95.9%)는 phenotypic 결과와 동일함을 확인했다. 새로 개발된 primer set를 이용한 결과는 universal primer set를 이용한 98개 균주(100%)의 결과와 동일함을 확인하였다. 개발된 16S rRNA gene PCR법은 임상미생물 검출 및 동정에서 신속성, 정확성, 그리고 검사 비용 절감의 장점을 가진다. 개발된 primer set는 병원성 미생물 동정에서 효율성을 확인했다. Broad-range and specific 16S rRNA gene PCR is used for detection and identification of bacterial pathogens in clinical specimens from patients with a high suspicion for infection. We describe the development of a broad-range and specific PCR primer, based on bacterial 16S rRNA, for use in routine diagnostic clinical microbiology services. The primers were designed by using conservative regions of 16S rRNA sequences from 10 strains. Ninety-eight clinical strains were isolated from clinical patient specimens. A total of 98 strains of bacteria were identified by phenotypic methods; PCR with newly designed primers and universal primers. All purified PCR products were sequenced using both forward and reverse primers on an automated DNA analyzer. In this study, we evaluated the usefulness of the newly designed primers and the universal primers for the detection of bacteria, and both these techniques were compared with phenotypic methods for bacteria detection. When we also tested 98 strains of clinical isolates with newly designed primers, about 778 bp DNA fragments were amplified and identified from all strains. Of the 98 strains, 94 strains (95.9%) correspond in comparison with phenotypic methods. The newly designed primers showed that the identities of 98 (100%) strains were the same as those obtained by universal PCR primers. The overall agreement between the newly designed primers and universal primers was 100%. The primer set was designed for rapid, accurate, and cheap identification of bacterial pathogens. We think the newly designed primer set is useful for the identification of pathogenic bacteria.

다용도 프라이머가 레진 시멘트와 수복재의 전단 결합 강도에 미치는 영향

김나홍,심준성,문홍석,이근우 대한치과보철학회 2012 대한치과보철학회지 Vol.50 No.2

연구 목적: 이 연구의 목적은 실란과 인산 모노머를 혼합한 프라이머인 Monobond plus (Ivoclar Vivadent, Schaan, Liechtenstein)를 사용했을 때 레진 시멘트와 수복물 간의 전단 결합 강도가 귀금속, 비귀금속, 글라스 세라믹과 지르코니아 네 가지 재료 모두에서 기존의 프라이머를 사용했을 때의 결합 강도와 비교하여 유의한지 평가하는 것이다. 연구 재료 및 방법: 디스크 모양(9 mm × 3 mm)의 귀금속(Argedent Euro) 시편 16개, 비귀금속(T-4) 시편 20개, 지르코니아(Cercon) 시편 20개, 글라스 세라믹(IPS e.max press) 시편 20개를 제작한 후 아크릴릭 레진(15 mm × 15 mm)에 포매하였다. 귀금속 시편에 airborne-particle abrasion을 시행하고 대조군으로 사용한 8개 시편에는 귀금속용 프라이머(Metal primer II)를, 나머지 8개 시편에는 Monobond plus를 도포하였다. 비귀금속 시편과 지르코니아 시편은 airborne-particle abrasion 후 각각 두 그룹으로 나누어 대조군 10개 시편에는 비귀금속 및 지르코니아용 프라이머(Alloy primer)를, 나머지 10개에는 Monobond plus를 도포하였다. 글라스 세라믹 시편은 4% 불산으로 부식한 후, 대조군 10개 시편에는 실란(Monobond-S)을, 나머지 10개에는 Monobond plus를 적용했다. 표면 처리된 시편 위에 디스크 형태(5 mm × 2 mm)로 레진 시멘트(Multilink N)를 위치시키고 중합하였다. 제작된 모든 시편을 열순환(5℃와 55℃, 1분씩 2060회)시킨 후 전단 결합 강도를 측정하였다. 전단 결합 강도의 유의차를 살펴보기 위해 Shapiro-Wilk test를 이용하여 모집단의 분포에 대한 검정을 하고 그 결과에 따라 Two sample t-test 또는 Mann-Whitney U test를 실시하였다(α=.05). 파절된 시편을 확대경으로 관찰하여 그 양상을 분류하였다. 결과: 귀금속과 글라스 세라믹 군에서는 두 프라이머간의 전단 결합 강도에 유의한 차이가 존재하지 않았으나(P> .05) 비귀금속 군과 지르코니아 군에서는 기존 프라이머(Alloy primer)를 사용했을 때 레진 시멘트와 수복물간의 전단 결합 강도가 Monobond plus를 사용한 군보다 통계적으로 유의하게 높았다(비귀금속 군 P=.004, 지르코니아 군 P=.001). 결론: 실란과 인산 모노머를 혼합한 다용도 프라이머는 귀금속과 글라스 세라믹 군에서는 기존의 프라이머를 대신하여 사용할 수 있을 것이다. 그러나 비귀금속과 지르코니아 세라믹에서는 10-MDP 프라이머에 비해 접착 강도가 낮아 기존 프라이머를 대신하여 적용하기 위해서는 좀 더 연구가 필요할 것이다. Purpose: The purpose of this study was to evaluate the difference in shear bonding strength between resin cements to dental materials when a universal primer (Monobond plus) was applied in place of a conventional primer. Materials and methods: Four groups of testing materials: gold alloy (Argedent Euro, n = 16), non precious metal (T-4, n = 20), zirconia (Cercon, n = 20) and glass ceramic (IPS e.max press, n = 20), were fabricated into discs, which were embedded in an acrylic resin matrix. The gold alloy specimens were airborne-particle abraded, 8 of the specimens were coated with Metal primer II, while the remaining 8 specimens were coated with Monobond plus. The non precious and zirconia specimen were airborne-particle abraded then, the control group received Alloy primer coating, while the other was coated with Monobond plus. Glass ceramic specimens were etched. 10 specimens were coated with Monobond-S and the remaining specimens were coated using Monobond plus. On top of the surface, Multilink N was polymerized in a disc shape. All of the specimens were thermal cycled before the shear bonding strength was measured. Statistical analysis was done with Two sample t-test or Mann-Whitney U test (α=.05). Results: There were no significant differences in bonding strength depending on the type of primer used in the gold alloy and glass ceramic groups (P>.05), however, the bonding strengths of resin cements to non precious metal and zirconia groups, were significantly higher when the alloy primer was used (P<.05). Conclusion: Within the limitations of this study, improvement of universal primers which can be applied to all types of restorations is recommended to precious metals and zirconia ceramics. But, the bond strengths of non precious metals and zirconia ceramics were significantly lower when compared to a 10-MDP primer. More research is needed to apply universal primers to all types of restorations.

일반 프라이머를 이용한 PCR의 식품원료 진위 판별에 적용

박용춘,진상욱,임지영,김규헌,이재황,조태용,이화정,한상배,이상재,이광호,윤혜성 한국식품위생안전성학회 2012 한국식품위생안전성학회지 Vol.27 No.3

In order to determine an authenticity of food ingredient, we used DNA barcode method by universal primers. For identification of animal food ingredients, LCO1490/HCO2198 and VF2/FISH R2 designed for amplifying cytochrome c oxidase subunit1 (CO1) region and L14724/H15915 for cytochrome b (cyt b) region on mitochondrial DNA were used. Livestock (cow, pig, goat, sheep, a horse and deer) was amplified by LCO1490/HCO 2198, VF2/FISH R2 and L14724/H15915 primers. Poultry (chicken, duck, turkey and ostrich) was amplified by LCO1490/HCO 2198 and VF2/FISH R2 primers. But, Fishes (walleye pollack, herring, codfish, blue codfish, trout,tuna and rockfish) were only amplified by VF2/FISH R2 primers. For plant food ingredients, 3 types of primers (trnH/psbA, rpoB 1F/4R and rbcL 1F/724R) have been used an intergenic spacer, a RNA polymerase beta subunit and a ribulose bisphosphate carboxylase region on plastid, respectively. Garlic, onion, radish, green tea and spinach were amplified by trnH/psbA, rpoB 1F/4R and rbcL 1F/724R. The PCR product sizes were same by rpoB 1F/4R and rbcL 1F/724R but, the PCR product size using trnH/psbA primer was different with others for plants each. We established PCR condition and universal primer selection for 17 item's raw materials for foods and determine base sequences aim to PCR products in this study. This study can apply to determine an authenticity of foods through making an comparison between databases and base sequences in gene bank. Therefore, DNA barcode method using universal primers can be a useful for species identification techniques not only raw materials but also processed foods that are difficult to analyze by chemical analysis.

세균의 Universal Primer를 이용한 균혈증의 진단

어영,이창훈,이미경,박일구,박덕우,윤갑준 대한임상병리학회 1998 대한임상병리학회지 Vol.18 No.2

다용도 프라이머로 전처리한 지르코니아에 대한 자가접착 레진 시멘트의 미세 전단 결합 강도

이임결(Lin-jie Li),이두형(Du-Hyeong Lee),권태엽(Tae-Yub Kwon),진명욱(Myoung-Uk Jin),이규복(Kyu-Bok Lee) 대한치과재료학회 2013 대한치과재료학회지 Vol.40 No.2

The objective of the study was to evaluate conventional and self-adhesive resin cements bonding to zirconia ceramic that were sandblasted and primed with universal primer. 8 sintered zirconia ceramic disks (Ceramill Zolid; Amann Girrbach, Austria) were air-abraded with 110 ㎛ Al₂O₃ at 2.5 bar pressure for 15s at a distance of 10 ㎜ and ultrasonic cleaned in 95% alcohol for 3min. All the specimens were applied by universal primer 60s and dried. The primed specimens were divided into 4 groups (n=10) according to the resin cement used: RelyX Unicem, Multilink Speed, MaxCem Elite) Panavia F2.0. The resin cement cylinders were stored in distilled water at 37℃ for 24h. Microshear bond strength (MSBS) test was measured with a universal testing machine. The means and standard deviations of microshear bond strength for RelyX Unicem, Multilink Speed, MaxCem Elite, Panavia F2.0 were 18.57 (4.80), 8.21 (4.95), 4.59 (3.14), 4.61 (2.80) MFa, respectively. Statistical analysis revealed that the MSBS of RelyX Unicem, Multilink Speed were significantly higher than MaxCem Elite, Panavia F2.0 (p<0.05). When self-adhesive resin cement bond to zirconia, universal primer plays a role of improving the bond strength to zirconia. It is recommended to use RelyX Unicem, Multilink Speed bond to zirconia which is primed with universal primer.

16S rDNA sequence에 대한 종특이성 primer를 이용한 중합효소연쇄반응증폭에 의한 Porphyromonas endodontalis의 동정에 관한 연구

엄승희,임성삼,배광식 大韓齒科保存學會 1999 Restorative Dentistry & Endodontics Vol.24 No.1

Development of a Novel Long-Range 16S rRNA Universal Primer Set for Metagenomic Analysis of Gastrointestinal Microbiota in Newborn Infants

( Hye Jin Ku ),( Ju Hoon Lee ) 한국미생물 · 생명공학회 2014 Journal of microbiology and biotechnology Vol.24 No.6

Metagenomic analysis of the human intestinal microbiota has extended our understanding of the role of these bacteria in improving human intestinal health; however, a number of reports have shown that current total fecal DNA extraction methods and 16S rRNA universal primer sets could affect the species coverage and resolution of these analyses. Here, we improved the extraction method for total DNA from human fecal samples by optimization of the lysis buffer, boiling time (10 min), and bead-beating time (0 min). In addition, we developed a new longrange 16S rRNA universal PCR primer set targeting the V6 to V9 regions with a 580 bp DNA product length. This new 16S rRNA primer set was evaluated by comparison with two previously developed 16S rRNA universal primer sets and showed high species coverage and resolution. The optimized total fecal DNA extraction method and newly designed long-range 16S rRNA universal primer set will be useful for the highly accurate metagenomic analysis of adult and infant intestinal microbiota with minimization of any bias.

Comparison of bond strengths of ceramic brackets bonded to zirconia surfaces using different zirconia primers and a universal adhesive

Lee, Ji-Yeon,Ahn, Jaechan,An, Sang In,Park, Jeong-won The Korean Academy of Conservative Dentistry 2018 Restorative Dentistry & Endodontics Vol.43 No.1

Objectives: The aim of this study is to compare the shear bond strengths of ceramic brackets bonded to zirconia surfaces using different zirconia primers and universal adhesive. Materials and Methods: Fifty zirconia blocks ($15{\times}15{\times}10mm$, Zpex, Tosoh Corporation) were polished with 1,000 grit sand paper and air-abraded with $50{\mu}m$ $Al_2O_3$ for 10 seconds (40 psi). They were divided into 5 groups: control (CO), Metal/Zirconia primer (MZ, Ivoclar Vivadent), Z-PRIME Plus (ZP, Bisco), Zirconia Liner (ZL, Sun Medical), and Scotchbond Universal adhesive (SU, 3M ESPE). Transbond XT Primer (used for CO, MZ, ZP, and ZL) and Transbond XT Paste was used for bracket bonding (Gemini clear ceramic brackets, 3M Unitek). After 24 hours at $37^{\circ}C$ storage, specimens underwent 2,000 thermocycles, and then, shear bond strengths were measured (1 mm/min). An adhesive remnant index (ARI) score was calculated. The data were analyzed using one-way analysis of variance and the Bonferroni test (p = 0.05). Results: Surface treatment with primers resulted in increased shear bond strength. The SU group showed the highest shear bond strength followed by the ZP, ZL, MZ, and CO groups, in that order. The median ARI scores were as follows: CO = 0, MZ = 0, ZP = 0, ZL = 0, and SU = 3 (p < 0.05). Conclusions: Within this experiment, zirconia primer can increase the shear bond strength of bracket bonding. The highest shear bond strength is observed in SU group, even when no primer is used.

Comparison of bond strengths of ceramic brackets bonded to zirconia surfaces using different zirconia primers and a universal adhesive

Ji-Yeon Lee,박정원,Jaechan Ahn,Sang In An 대한치과보존학회 2018 Restorative Dentistry & Endodontics Vol.43 No.1

Objectives The aim of this study is to compare the shear bond strengths of ceramic brackets bonded to zirconia surfaces using different zirconia primers and universal adhesive. Materials and Methods Fifty zirconia blocks (15 × 15 × 10 mm, Zpex, Tosoh Corporation) were polished with 1,000 grit sand paper and air-abraded with 50 µm Al2O3 for 10 seconds (40 psi). They were divided into 5 groups: control (CO), Metal/Zirconia primer (MZ, Ivoclar Vivadent), Z-PRIME Plus (ZP, Bisco), Zirconia Liner (ZL, Sun Medical), and Scotchbond Universal adhesive (SU, 3M ESPE). Transbond XT Primer (used for CO, MZ, ZP, and ZL) and Transbond XT Paste was used for bracket bonding (Gemini clear ceramic brackets, 3M Unitek). After 24 hours at 37°C storage, specimens underwent 2,000 thermocycles, and then, shear bond strengths were measured (1 mm/min). An adhesive remnant index (ARI) score was calculated. The data were analyzed using one-way analysis of variance and the Bonferroni test (p = 0.05). Results Surface treatment with primers resulted in increased shear bond strength. The SU group showed the highest shear bond strength followed by the ZP, ZL, MZ, and CO groups, in that order. The median ARI scores were as follows: CO = 0, MZ = 0, ZP = 0, ZL = 0, and SU = 3 (p < 0.05). Conclusions Within this experiment, zirconia primer can increase the shear bond strength of bracket bonding. The highest shear bond strength is observed in SU group, even when no primer is used.

Effect of pre-curing of two universal adhesives on the shear bond strength of resin cement to zirconia

Ga-Eun Son(손가은),Tae-Yub Kwon(권태엽),Young Kyung Kim(김영경) 대한치과재료학회 2019 대한치과재료학회지 Vol.46 No.1

This study aimed to investigate whether pre-curing the universal adhesives affect the resin bonding to zirconia ceramic. Two commercial universal adhesives (All-Bond Universal, ABU; Single Bond Universal Adhesive, SBU) were tested in comparison with a conventional zirconia primer (Z-Prime Plus, ZPP). Air-abraded zirconia specimens were divided into six groups, to which different priming agents were applied with or without photo-polymerization. After resin cylinders were built on the primed zirconia surface using a resin cement (Duo-Link), all bonded specimens were stored in water at 37℃ for 24 hours and half of them (n = 12) were additionally thermocycled 5,000 times. A shear bond strength test was performed at a crosshead speed of 0.5 mm/minute and failure modes were assessed using an optical microscope at 25× magnification. Before thermocycling, the light-cured specimens of ABU and SBU showed higher bond strengths than the uncured ones of the materials (p<0.05), whereas no significant difference was found between the pre-cured and uncured specimens of ZPP (p > 0.05). After thermal aging, there were significant decreases in the bond strength for all specimens treated with the universal adhesives (p<0.05). However, ZPP produced no significant difference after aging (p > 0.05) and attained higher bond strength values for both the pre-cured and uncured specimens when compared to the two universal adhesives (p<0.05). Adhesive failures were predominant in all test groups and thermocycling resulted in the higher incidences of adhesive failures for the uncured groups, which was not shown in the pre-cured groups.